The increase in the half-life in the blood of a therapeutic active ingredient has advantages, including fewer administrations being necessary to gain the desired therapeutic effect. This reduction in the number of administrations is of special importance in drugs for parenteral administration, most especially in those for intravenous use and of special relevance to long-term medications such as, for example, those for the treatment of chronic disorders.
The current tendency is, as far as possible, to administer active ingredients by routes which do not need intravenous access, because of complexity and inconvenience for the patient when this method is used. However, there is a series of active ingredients for which there is at present no alternative to intravenous administration. Included in these are active ingredients of great size and complexity, such as biological or biotechnological products, which include proteins and hormones.
One example of a chronic therapeutic condition where the repeated intravenous administration of complex active ingredients is necessary is haemophilia. Haemophilia is a hereditary disease featuring the appearance of internal and external bleeding due to the total or partial deficiency of proteins related to the clotting of blood. Haemophilia A features a deficiency of clotting Factor VIII, which impedes the normal generation of thrombin, making it difficult for the blood to clot normally as a result. In the case of haemophilia B, the deficiency of Factor IX causes a similar clinical state.
For the treatment of haemophilia, the first therapeutic option consists of replacing the absent protein (FVIII or FIX) by the administration of a therapeutic concentrate containing this factor. Another therapeutic option to obtain correct haemostasis in haemophilia is the administration of FVIIa, which has the ability to generate thrombin in the absence of FVIII or FIX. However, this type of treatment is usually limited to cases where treatment with FVIII or FIX is problematic or has proved ineffective, such as for example in patients who have had an inhibitory immunological response to these active ingredients. To date, none of these products has been successfully administered by any method of administration except intravenous, given its structural complexity and low epithelial permeability.
Therefore, patients affected by haemophilia require intravenous administrations repeated with a frequency determined by its half-life in the plasma. In the case of FVIII the half-life is about twelve hours. This implies, according to the monograph of the World Federation of Haemophilia (Casper, C K, Hereditary Plasma Clotting Factor Disorders and Their Management 5th ed. WFH, Sam Schulman Ed., 2008), that for a primary prophylaxis regime, i.e., for the prevention of bleeding in children without articular damage a dose of about 20 U/kg every 48 hours is used, sufficient to maintain a level of plasma FVIII of more than 1% of the normal value. Essentially, this treatment changes a person with severe haemophilia into one with slight or moderate haemophilia. In the case of FIX, the half life is about 26 hours, so that for primary prophylaxis doses of about 40 U/kg twice a week can be administered in order to maintain a minimum level of 1%.
It has to be taken into account that prophylaxis from an early age (about age one year or at the start of crawling) is the standard of care required in order to avoid articular damage in cases of severe haemophilia.
Consequently, haemophilia is a clear example where an increase in the half-life of the active ingredient would provide a substantial improvement in the patient's quality of life, as it would reduce the number of intravenous administrations, especially difficult in children of a young age.
Other examples of long-term treatments with intravenous administration products are for example, the use of immunoglobulins (IgG) in primary immunodeficiencies and the use of antithrombin III (AT) and alpha-1 antitrypsin (AAT) in congenital deficiencies.
There are numerous technological approaches aiming at extending the plasma half-life of these types of active ingredients. One of the most studied has been the derivatisation of proteins with compatible polymers, as is the case of polyethylene glycol (PEG). This technology consists of the practice of carrying out a chemical reaction to join PEG chains covalently to protein amino acids. This technique has proved useful in the case of hormones and peptide chains of small size, such as interferon, since for compounds of this type the principal mechanism of elimination is renal clearance, easily controllable by a simple increase in size (Bailon Pascal et al, Bioconjugate Chem. 2001, 12, 195-202). However, it is still to be decided whether it can be used in more complex active ingredients, as they are based on the external modification of the protein structure to be treated. In addition, covalent bonds of this type with the protein considerably reduce the biological activity of the treated hormone or protein.
Another alternative to modify the half-life has been the addition or modification of the sugar residues naturally present in proteins or hormones (Perlman Signe et al, The Journal of Clinical Endocrinology & Metabolism 88 (7): 3227-3235, 2003). This procedure claims to alter the protein, by modifying its recognition by the receptors involved in its degradation. However, the inherent risks of this alteration are obvious, given the high immunogenic potential of the glycosylations present in the proteins.
A third line of action has been to obtain chimeric proteins where the active sequence of a protein of interest is expressed, bonded to sequences of plasma proteins which have a considerable half-life, as is the case of albumin or fragments of immunoglobulins (Dennis, Marks S. et al, The Journal of Biological Chemistry vol. 277, No. 38, Issue of September 20, pp. 35035-35043, 2002). However, this technology has as its principal disadvantage, in addition to the expected immunogenicity associated with exposing patients to proteins not present in nature, loss of efficacy of the protein upon the modification of its structure in such a dramatic way.
Another possibility investigated to extend the half-life of complex active ingredients has been the co-administration of the product with a liposome stabilised with PEG. This technique is based on the affinity of the active ingredient for PEG, which allows a reversible association between the protein and the liposome. This transitory association must provide an increase in the half-life of the active protein ingredient, since liposomes stabilised with PEG stay in circulation for a long time. However, it has not been possible to corroborate this hypothesis in practice, as this system has proved to be ineffective in extending the half-life of FVIII in haemophilia patients (Powell J. S et al, Journal of Thrombosis and Haemostasis, 6: 277-283, 2007).
To date, no system amongst those previously described has been able to significantly modify the half-life, with the exceptions described where the introduction of structural modifications and alterations make their application unviable or very complex for the treatment of human pathologies.
The controlled release of therapeutic agents encapsulated in biodegradable polymeric microspheres has been extensively studied. The microencapsulation of the active ingredient in biodegradable polymers allows the release of the drug to be controlled. This approach has recently been applied in controlled release formulations for subcutaneous use based on derivatives of lactic and glycolic acids. These formulations have been used successfully in the encapsulation of a wide variety of active ingredients, including cytostatics, anti-inflammatories, peptides and hormones, inter alia (Tamilvanan S. et al, PDA Journal of Pharmaceutical Science and Technology, vol. 62, No. 2, March-April 2008 pp. 125-154).
Pankaj (U.S. Pat. No. 5,417,982) describes the use of lactic and glycolic acid microspheres for the controlled release of hormones by oral administration. Although Pankaj describes the possibility of obtaining an injectable product, it is very unlikely that this invention can be administered intravenously, given the requirements of this method of administration, and in any case, this invention does not anticipate the use of alginates for this purpose.
Sivadas (Sivadas Neeraj et al, International Journal of Pharmaceutics 358 (2008) pp. 159-167) describes the use of different polymers, including hydroxypropyl cellulose, chitosan, hyaluronic acid, gelatine, ovalbumin and glycolic polylactic acid, as vehicles for the encapsulation of proteins for their administration by inhalation.
One disadvantage of the use of lactic and glycolic acid derivatives is the need to make the preparations in the presence of organic solvents, some of them of known toxicity, such as polyvinyl alcohol, which exhibit incompatibilities with the conservation of the biological activity of complex active ingredients such as proteins and hormones.
The use of these polymers also results in highly hydrophobic particles, which, as is discussed below, are rapidly eliminated from the circulation by cellular uptake mechanisms. An additional disadvantage is the creation of a locally very acid environment around the particle at the time of its dissolution and, therefore, at the time when the active ingredient is released. This is due to the fact that the polymer decomposes in lactic acid and glycolic acid, which creates an extremely acidic environment around the particle in dissolution. It is this acid environment which can damage sensitive active ingredients and particularly those which have complex amino acid structures with labile biological activity.
Alginates have many applications in the food and pharmaceutical industries and in the chemical industry in general. This wide variety of applications is defined by their hydrocolloid property, i.e., their ability to hydrate themselves in water so as to form viscous solutions, dispersions or gels. This feature gives alginates unique properties as thickening agents, stabilising agents, gelling agents and film formers.
One area where the properties of alginates have been widely exploited has been in the encapsulation of active ingredients in particular in order to improve their solubility, or to assist the administration of drugs (Tønnesen, Hanne Hjorth et al, Drug Development and Industrial Pharmacy, 28(6), 621-630 (2002)) by various routes. Amongst these is the use of oral administration given the mucoadhesive properties of alginate. The subcutaneous method has also been examined. However there is no history of intravenous use due to the strict requirements of this route of administration.
For example, Benchabane (Benchabane, Samir et al, Journal of Microencapsulation, September 2007; 24(6): pp. 565-576) describes the use of alginates in the production of albumin microcapsules by “spray-drying” for oral administration. In a similar antecedent, Coppi (Coppi, Gilberto et al, 2001, Drug Development and Industrial Pharmacy, 27(5), pp. 393-400) demonstrates the formation of microspheres crosslinked with calcium and chitosan for the oral administration of proteins. In both cases, alginate acts as a protector of protein against the proteolytic degradation which occurs naturally during gastric digestion.
Further, Mladenovska (Mladenovska, K., International Journal of Pharmaceutics 342 (2007) pp. 124-136) describes obtaining microparticles of alginate/chitosan for colonic administration.
Sivadas (Sivadas Neeraj et al, International Journal of Pharmaceutics 358 (2008) pp. 159-167) also mentions the use of alginates as a vehicle for the encapsulation of proteins for administration by inhalation.
Apart from the direct administration of active ingredients, alginates have also been suggested as vehicles for the administration of complex therapeutic forms. For example, in patent WO 2006/028996 A2 the use of alginate and Emulsan for the transport of detoxifying agents of bacterial toxins is described.
Another example is the use of alginate in the encapsulation of multivesicular liposomes (Dai, Chuanyun, et al, Colloids and Surfaces B: Biointerfaces 47 (2006) pp. 205-210) or live cells (European Patent, publication number: 2 159 523). In this case, the administration of live cells has as its objective their application in regenerative medicine or gene therapy (WO 2007/046719 A2; Peirone, Michael et al, J. Biomed. Mater. Res. 42, pp. 587-596, 1998; García-Martín, Carmen et al, The Journal of Gene Medicine, J Gene Med 2002; 4: pp. 215-223). Curiously, García-Martín (García-Martín, Carmen et al, The Journal of Gene Medicine, J Gene Med 2002; 4: pp. 215-223) describes the possible application of the administration of genetically modified live cells for the treatment of haemophilia A, exemplifying the medical relevance of the problem. In this case, alginate microcapsules which contain live cells are implanted intraperitoneally by the introduction of a catheter. In this case, both the objective of the treatment and the method of administration—non-intravenous—are radically far from the present invention.
In spite of this wide experience in the use of polymers for the encapsulation of complex active ingredients, such as proteins, there are no references which can resolve the problems associated with the intravenous administration of these products. As Wong et al describe (Wong, Joseph et al, Advanced Drug Delivery Reviews 60 (2008) pp. 939-954) there are only three approved products which use particle suspensions for their intravenous administration. None of them include the use of alginates in their composition. In all cases, an increase in half-life is not sought, but an improvement in the solubility of the product.
The difficulty of effectively administering microparticles intravenously can be expressed in (a) the basic aspects of design of the product, such as the size of the particle and distribution, absence of organic solvents, and also the homogeneity, viscosity and “syringeability” of the suspension—understanding as “syringeability” the ease of suction and injection of the product; (b) the technical aspects of production and preparation on an industrial scale, such as the uniformity of the dose, the unwanted crystallisation of salts in the case of products obtained by solvent precipitation, the sterility and apyrogenicity of the product; and (c) biological aspects, such as the non-deliberate alteration of the pharmacokinetic and pharmacodynamic profile, alteration of the biodistribution, the bioaccumulation of the polymer, phagocytic activation, toxicity and effects of embolisation or activation of the complement.
In this connection, one of the most significant problems in the development of these products is its fast clearance by the mononuclear phagocyte system (MPS), previously called reticuloendothelial system (RES), which includes all the cells derived from the monocytic precursors of the bone marrow, the monocytes of the peripheral blood and the macrophages or histiocytes of the various organs and tissues. Amongst the latter must be mentioned, because of their importance in the clearance of microparticles in plasma, the Küpfer cells of the liver and the macrophages distributed in the spleen and the bone marrow (Passirane, Catherine et al, Pharmaceutical Research, Vol. 15, No. 7, 1998 pp. 1046-1050).
It has been widely described that after the intravenous administration of nano- or micro-particles they are rapidly opsonised by the proteins of the plasma. These proteins absorbed in their surface induce recognition and uptake by the MPS cells (Passirane, Catherine et al, Pharmaceutical Research, Vol. 15, No. 7, 1998 pp. 1046-1050).
A similar effect has been observed in liposomes (Ishida, Tatsuhiro et al, Journal of Controlled Release 126 (2008) pp. 162-165), where a phenomenon known as Accelerated Blood Clearance (ABC) has been described. Both in the case of polymeric microparticles and in that of liposomes, the opsonisation phenomena are also directly related to the activation of the complementary system (Ishida, Tatsuhiro et al, Journal of Controlled Release 126 (2008) pp. 162-165; Koide, Hiroyuki et al, International Journal of Pharmaceutics 362 (2008) pp. 197-200).
In practice, this phagocytosis phenomenon prevents the development of drugs with an extended half-life based on microparticles administered intravenously, since the increase in size associated with encapsulation does not just increase but on occasions causes accelerated degradation. Obviously, this phenomenon is only observed by means of in vivo experimentation, which involves studies of pharmacokinetics in animals.
The relationship between this clearance via phagocytosis and the size of the particle has been widely documented. Champion (Champion, J A, Pharm Res. 2008 August; 25(8): 1815-21. Epub 2008 Mar. 29) specifically describes the relationship between the phagocytosis experienced by polymeric microparticles and their size, observing a maximum effect between 2-3 μm. Other features which define the uptake of microparticles by the MPS in vivo are the hydrophobicity of the particles and their Zeta Potential (Z Potential) (Szycher, Michael, High Performance Biomaterials: A Comprehensive Guide to Medical and Pharmaceutical Applications, published by CRC Press, 1991 ISB 0877627754, 9780877627753, 812 pages).
Z Potential is a property of the particles. Specifically, disperse particles tend to become electrically charged by the adsorption of ions from the external phase, or by ionisation of functional groups on their own surface. One consequence of this is that a layer of counterions called the Stern layer will appear back to back with the particle in the environment of a negatively charged dispersed particle. A diffused layer appears on said stern layer featuring the presence of mobile charges (of both signs) which will counteract the charge of the particle, as a function of the distance to the same. Z Potential is what we call the difference in potential between the layer of counterions and the point of electrokinetic neutrality.
Z Potential values are crucial for the stability of the majority of dispersed systems, since the latter will regulate the degree of repulsion between dispersed particles of similar charge, preventing said particles from coming too close to one another and the forces of inter-particle attraction, caused by the coalescence phenomena, from becoming predominant. As regards the Z potential, it has been disclosed (Szycher, Michael, High Performance Biomaterials: A Comprehensive Guide to Medical and Pharmaceutical Applications, published by CRC Press, 1991 ISB 0877627754, 9780877627753, 812 pages) that partially negative Z potentials close to 0 reduce phagocytosis.
Moreover, hydrophobicity also assists the opsonisation and uptake of the particles. This is of particular interest, since particles derived from polylactic and glycolic acids are, for example, highly hydrophobic.
One approach achieved to extend the half-life in plasma of microparticles and liposomes was the introduction, onto the surface thereof, of charged polymers which are able to modify their charge and generate a hydrophilic surface layer to protect them from opsonisation and phagocytosis. Amongst them is the use of polyethylene glycol (PEG) (Ishida, Tatsuhiro et al, Journal of Controlled Release 126 (2008) 162-165; Owens III, Donald E et al, International Journal of Pharmaceutics, volume 307, Issue 1, 3 Jan. 2006, Pages 93-102) or heparin (Passirane, Catherine et al, Pharmaceutical Research, Vol. 15, No. 7, 1998 pp. 1046-1050).
This approach complicates and makes difficult the development of a pharmaceutical product because of the increase in the complexity of the system. In addition, as has been previously discussed, the use of PEG-liposomes has proved to be ineffective in extending the half-life of a complex protein such as FVIII (Powell J. S et al 2007, Journal of Thrombosis and Haemostasis, 6: pp. 277-283).
In the case of microparticles, in order to obtain a viable product for intravenous administration it would be necessary to have hydrophilic particles with a suitable combination of size and Z potential.
Terrence (European Patent, Publication Number: 2 286 040, European Application Number: 00973477.3) describes the use of polymers as a system of administration capable of increasing the half-life of the active encapsulated ingredients. For this purpose, this invention requires the use of (1) a first water-soluble polymer, (2) at least one anionic polysaccharide as first complexing agent and (3) a divalent cation as a second complexing agent. As has been observed, the invention mentioned is technically complex and difficult to use in practice. In contrast, in the present invention the controlled release of the active ingredient is achieved with far simpler microparticles, which involve the use of a single polymer that possesses all the properties necessary for its application. Furthermore, Terrence's invention does not demonstrate the compatibility of its preparation for intravenous use by size, or explain or illustrate how to avoid cellular phagocytosis.
Alginate, unlike other polymers with PLA or PLGA, is hydrophilic. Particles generated in the present invention have been shown to have partially negative Z potentials sufficient to prevent the aggregation of particles, but neutral enough to provide a low opsonisation profile.
The maximum sizes of particle acceptable for intravenous administration are around 5 μm. This is demonstrated by the existence of registered drugs which use albumin marked for diagnosis by ultrasounds (Optison, data sheet 28) with an average size of 3.0-4.5 μm.
Alginate is biocompatible, and has been used extensively for oral administration in humans, given its wide use in the food industry. When injected intravenously as a non-particulate polymer, it is eliminated in a biphasic form with half-lives of 4 and 22 hours (Hagen, A. et al, European Journal of Pharmaceutical Sciences, Volume 4, Supplement 1, September 1996, pp. 100-100 (1)) without adverse effects being observed. Alginate is eliminated via urine.
In addition, the fact that it is a water-soluble polymer assists its compatibility with complex proteins, as these latter are its natural solvent.
The present invention relates to a composition comprising microparticles of alginic acid or its pharmaceutically acceptable salts by which a controlled release is achieved, and achieves an increase in the half-life of the active ingredients administered intravenously, and results in a lower frequency of application and achieves more stable levels of active ingredient in the blood, thus potentially reducing the peaks and troughs typical in the concentration of the active ingredient, which occur as a result of the periodical infusion of the same.
The present invention describes hydrophilic microparticles of alginate with a combination of size suitable for intravenous infusion and physio-chemical characteristics suitable for preventing the rapid phagocytosis of the same, allowing a controlled release of complex active ingredients.